The intracellular transport and targeted delivery of cell surface and secreted proteins is a fundamental process in all eukaryotic cells. The machinery that recruits cargo proteins from the lumen and membrane of secretory organelles, deforms the membrane for transport vesicle formation, and targets vesicle fusion to the appropriate location has been described for several transport steps and shown to be well conserved among eukaryotes. The specificity of cargo sorting and targeting demands unique regulatory and structural proteins at each transport step. The yeast Saccharomyces cereviseae has been invaluable in identifying and characterizing many of these structural and regulatory proteins and is a well-established model system in the study of the secretory process. However, the branching of transport routes in both yeast and mammalian cells has complicated studies of the late (post-Golgi) secretory pathway, and the molecular machinery required for exocytic cargo sorting and exit from the Golgi and endosomes is largely unknown. Because cargo can be sorted away from a blocked route and secreted by an alternate route, mutants defective in only one route from the Golgi do not exhibit an easily screened secretory phenotype and are therefore difficult to isolate. We are approaching this problem by conducting screens with a mutant yeast strain that is already defective in one of the two known exocytic routes, so that interfering with the remaining route should cause a complete secretory block that is easy to detect. We have successfully used this strategy in a classical genetics (mutant screen) approach; however, a "chemical genetics" approach promises to be more efficient in identifying the desired genes an should in addition, yield chemical compounds with potential use in therapeutics or as a research tool. We therefore propose to use a high-throughput, cell-based screen of a chemical library to identify compounds (and subsequently, their targets) that interfere specifically with one branch of the exocytic pathway. [unreadable] [unreadable]